Alzheimer's Disease (AD) is a progressive, neurodegenerative disease characterized by cognitive and behavioral changes which include: (a) memory loss; (b) language deterioration; (c) impaired visual-spatial skills; (d) poor judgment; (e) indifferent attitude and (f) aimless, unpredictable behavior. Although AD usually begins after age 60, its onset may occur as early as age 40. AD first appears as memory decline. As the disease progresses over several years, cognition, personality, and the ability to function are all impaired or destroyed. Confusion and restlessness may also occur. The type, severity, sequence, and progression of mental changes vary widely among AD patients. Some people have the disease for only the last 5 years of life, while others may have it for as many as 20 years. The most common cause of death in AD patients is infection.
There is no known etiology or cure for AD and no way to slow the progression of the disease. For some people in the early or middle stages of the disease, medication such as tacrine may alleviate some cognitive symptoms. Also, some medications may help control behavioral symptoms such as sleeplessness, agitation, wandering, anxiety, and depression. These treatments are aimed solely at making the patient more comfortable and do nothing to slow the progression of the underlying disease.
As such, there is much ongoing research that is aimed at the identification and development of new therapeutic agents which can at least slow, if not reverse, the progression of AD. An important step in the identification of therapeutic agents for AD would be the development of a non-human animal model of the disease, as such an animal model would serve as an invaluable tool for screening and testing potential therapeutic agents.
The only non-human animals that naturally develop AD pathological features are aged non-human primates. However, primates are expensive, difficult to use, and require a significant period of time prior to developing AD. These factors make the use of primates as AD animal models prohibitive.
Apolipoprotein E (apoE) is a 34,000 molecular weight protein which is the product of a single gene on chromosome 19. Human ApoE exists in three major isoforms designated apoE2, apoE3 and apoE4. The different isoforms result from amino acid substitutions at amino acid residue positions 112 and 158. The common isoform, apoE3, has a cysteine residue at position 112 and an arginine residue at position 158. The apoE4 isoform differs from apoE3 only at position 112, which is an arginine residue. The apoE4 isoform has been associated with neurological disorders such as AD, with poor outcome following stroke or traumatic head injury, accelerated progression of multiple sclerosis, and with certain cardiovascular disorders.
Human apoE4 exhibits domain interaction due to the presence of an Arg-112, together with an Arg-61 and a Glu-255, the latter two amino acids forming a salt bridge. ApoE4 domain interaction is predicted to represent a key factor responsible for its association with both heart disease and neurological disorders. Like many non-human animals, mouse apoE contains the equivalent of Arg-112 and Glu-255, but lacks the critical Arg-61 required for domain interaction; instead, mouse apoE, as well as apoE of at least nine other species, contains Thr-61. Weisgraber (1994) Adv. Protein Chem. 45:249–302.
ApoE contains two structural domains: an amino-terminal and a carboxyl-terminal domain. Weisgraber (1994) Adv. Protein Chem. 45:249–302. Each domain is associated with a specific function. The amino terminal domain contains the lipoprotein receptor binding region and the carboxy-terminal domain contains the major lipid-binding elements. The two domains interact with each other in an isoform-specific manner such that amino acid substitutions in one domain influence the function of the other domain, a phenomenon referred to as domain interaction.
Domain interaction is responsible for the preference of apoE4 for very low density lipoproteins (VLDL) contrasted with the preference of apoE3 for high density lipoproteins (HDL). The specific amino acid residues in apoE4 that are involved in this interaction have been identified: arginine-61 in the amino-terminal domain and glutamic acid-255 in the carboxyl-terminal domain. Dong et al. (1994) J. Biol. Chem. 269:22358–22365; and Dong and Weisgraber (1996) J. Biol. Chem. 271:19053–19057. In addition to differences in lipoprotein preference, apoE4 differs from apoE3 in its interaction with other proteins. For example, tau, a protein found in neurofibrillary tangles, interacts in vitro with apoE3, but not with apoE4.
ApoE4 transgenic animals expressing human apoE isoforms have been reported, including transgenic mice in which the endogenous mouse apoE gene is ablated or inactivated, and gene-targeted mice in which a human apoE coding sequences is stably integrated into the mouse apoE locus. See, e.g., U.S. Pat. Nos. 6,046,381; and 5,767,337. In the transgenic animal models, the human apoE-encoding transgene is under transcriptional control of control elements that are part of the transgene. Furthermore, the transgene inserts at essentially random sites in the genome. Although in the gene-targeted mice, expression of apoE is under the control of endogenous control elements that normally control apoE gene expression, such as tissue-specific elements and promoter elements, there is a potential for species effects.
In view of the foregoing, there is a need in the art for improved animal models of human apoE4 activity. The present invention addresses this need.
Literature
U.S. Pat. Nos. 5,767,337 and 6,046,381 relate to transgenic mice expressing human apoE4. U.S. Patent disclosing other transgenic AD animal models include: U.S. Pat. Nos. 5,777,194; 5,720,936; 5,672,805; 5,612,486; 5,602,309; and 5,387,742. Dong and Weisgraber (1996) J. Biol. Chem. 271:19053–19057; and Dong et al. (1994) J. Biol. Chem. 269:22358–22365 relate to apoE4 domain interaction. Baum et al. (2000) Microsc. Res. Tech. 50(4):278–281 review apoE isoforms in Alzheimer's disease pathology and etiology.